Compositions for Immunotherapy

ABSTRACT

The present invention relates to compositions which can be used in immunotherapy and especially to compositions which can be used in immunotherapy for mammals suffering from a peanut allergy. The present invention further relates to the use of the present compositions for the treatment of a mammal suffering from an allergy by immunotherapy and the use of the present compositions in a prophylactic treatment for desentizing the immune system of a mammal for an allergen. Specifically, the present invention relates compositions suitable for immunotherapy comprising an allergen, wherein substantially 100% of said allergen in said composition is complexed with aluminum. The allergen, e.g. peanut kernel protein extract, is preferably reduced and alkylated in these compositions.

The present invention relates to compositions which can be used inimmunotherapy and especially to compositions which can be used inimmunotherapy for mammals, such as human mammals, suffering from peanutallergy. The present invention further relates to the use of the presentcompositions for the therapeutic treatment for desentizing the immunesystem of a mammal suffering from an allergy by immunotherapy and theuse of the present compositions in a prophylactic treatment of a mammalwith high predisposition to develop a certain allergy.

Allergen immunotherapy, also termed hyposensitization therapy,immunologic desensitization, hyposensibilization, or allergen-specificimmunotherapy, is a form of immunotherapy for allergic disorders inwhich the patient is vaccinated with increasingly larger doses of anallergen, i.e. the substance, or substances, to which they are allergic,with the aim of inducing immunologic tolerance.

Allergen specific immunotherapy is the only treatment strategy whichtreats the underlying cause of an allergic disorder. It is a highlycost-effective treatment strategy and results in an improved quality oflife.

Immunotherapy has been shown to produce long-term remission of allergicsymptoms, to reduce severity of associated allergic response, as well toreduce the chances of new sensitizations to allergens developing.Immunotherapy aims to modulate the immune system's response toallergens.

Immunotherapy generally encompasses repeated exposure to a specificallergen via, for example, sublingual or subcutaneous routes, therebyproviding a desensitization of the allergic patient to the allergen andthus a reduction in allergic symptoms and use of symptomatic basedtreatments.

The exact mechanism underlying immunotherapy is not fully known but itis accepted that immunotherapy leads to alteration of the immuneresponse to an allergen. The modification at least comprises a change inIgE synthesis and the production of IgE blocking antibodies reducing theallergic response of the immune system to specific allergens. Also anincrease in conversion of Th2 to Th1/T regulatory cells is observed. Ata molecular level, part of the underlying mechanism relies on thepreferential induction of allergen-specific IgG to neutralize anallergen and a reduction of allergen-specific IgE.

Immunotherapy generally involves exposing an allergic patient to lowdoses of an allergen. The dose is gradually increased on a regular, forexample weekly, basis, until a “maintenance” dose is reached. Thistranslates in approximately four months of weekly injections to reachthe maintenance dose. Once the maintenance dose is reached, theinjections are administered less often, for example once per month for afew years. Generally, the longer the treatment and the higher the dose,the greater the therapeutic benefit.

After successful completion of immunotherapy, long-term protection canbe expected for a period of 3 to 5 years or more. Therapy can berepeated should symptoms begin to return or if the individual becomesexposed to new allergens that were not included in the previoustreatment regimen.

Peanuts are one of the most common foods responsible for food-inducedallergy. A curative treatment for peanut allergy is not yet available.Specific immunotherapy (SIT) using aqueous peanut extract displayed anincreased tolerance to oral ingestion of peanuts. However, as reportedby Nelson et al. (J. Allergy Clin. Immunol. 1997 June;99(6 Pt1):744-51), aqueous peanut extracts resulted in unacceptable systemicreactions, even during the maintenance injections. Accordingly, theNelson et al. concluded: “For clinical application of this method oftreatment, a modified peanut extract is needed.”

Considering the clinical relevance of immunotherapy, there is acontinuous need in the art for compositions suitable for immunotherapyand especially immunotherapy effective against peanut allergy. Aperquisite for these compositions is that the compositions need to be,besides providing an alteration of the immune response upon exposure toan allergen, safe, i.e. the compositions must not trigger an allergicreaction and, in the most severe case, an anaphylactic shock.

Considering the above need in the art, it is an object of the presentinvention, amongst other objects, to provide compositions suitable forimmunotherapy and especially immunotherapy directed to peanut allergy.

This object of the present invention, amongst other objects, is met by acomposition suitable for immunotherapy as defined in the appendedclaims.

Specifically, this object of the present invention, amongst otherobjects, is met, according to a first aspect, by a composition suitablefor immunotherapy comprising an allergen, wherein substantially 100% ofsaid allergen in said composition is complexed with aluminum.

Within the context of the present invention, an allergen is defined asan antigen capable of stimulating a hypersensitivity reaction in atopicmammals through immunoglobulin E (IgE) responses. Most mammals mountsignificant immunoglobulin E responses only as a defense againstparasitic infections. However, some mammals may respond to many commonenvironmental antigens. This hereditary predisposition is alsodesignated atopy. In atopic mammals, non-parasitic antigens stimulateundesired IgE production, resulting in hypersensitivity or allergy.

Common allergens include antigens found in animal products such as Fel d1 (cat allergy), fur and dander, cockroach calyx, wool and dust miteexcretion; drugs such as penicillin, sulphonamides, and salicylates;foods such as celery and celeriac, corn or maize, eggs (typically eggwhite), fruit, pumpkin, egg-plant, legumes, beans, peas, peanuts,soybeans, milk, seafood, sesame, soy, tree nuts, pecans, almonds, andwheat; insect stings such as bee sting venom, wasp sting venom, andmosquito stings; mold spores; latex; metal; and plant pollen such asgrasses and tree pollen.

Within the context of the present invention, the terms “allergen”,“allergens”, “antigen” and “antigens” are used interchangeably unlessindicated otherwise.

The present inventors have surprisingly found that when substantially100%, such as more than 99%, of an allergen in a preparation iscomplexed, or conjugated with, or bound to aluminum, no clinicallyrelevant allergic reactions, i.e. mast cell-mediated systemic responses,are observed although the aluminum complexed allergen is still capableof inducing an IgG response thereby providing a composition especiallysuitable to be used in immunotherapy.

Mast cell-mediated systemic responses can be readily measured by alowering of the body temperature after exposure to an allergen.

According to a preferred embodiment of this first aspect of the presentinvention, the present aluminum complexed, bound, or conjugated allergenis a peanut kernel protein extract, preferably a peanut kernel proteinextract being modified by reduction and subsequent alkylation.

The present peanut kernel protein extract can be obtained by a) grindingpeanuts for providing a peanut powder; b) incubating the peanut powderin acetone during 30 minutes using 5 grams peanut powder per 50 mlacetone for providing a defatted peanut powder; c) drying the defattedpeanut powder; d) suspending the dried peanut powder in a buffer with apH between 7 and 9; and e) isolating the resulting supernatant of step(d) thereby providing a peanut kernel protein extract.

Reduction of the present peanut extract can be provided by contactingthe extract with one or more reducing agents chosen from the groupconsisting of 2-mercaptoethanol (β-ME), dithiothreitol (DTT),dithioerythritol, cysteine, homocystein, tributylphosphine, sulfite,tris(2-carboxyethyl) phosphine (TCEP), sodium (cyano) borohydride, lye,glutathione, E-mercapto ethylamine, thioglycollic acid, methyl sulfide,and ethyl sulfide.

Subsequent alkylation of the present extract can be provided bycontacting the reduced extract with one or more alkylating agents chosenfrom the group consisting of N-ethylmalimide, cystamine, iodoacetamide,iodoacetic acid, alkylhalogenides; alkylsulfates; alkenes, preferablyterminal alkenes (H₂C)=C(H)—R, and enzymes.

According to another preferred embodiment of this first aspect of thepresent invention, the present a peanut kernel protein extract comprisesat least the major peanut allergens Ara h1, Ara h2 and Ara h6.

Peanut allergenic protein Ara h1 was described as a 63.5 kDa proteinoccurring naturally in a trimeric form of approximately 180 kDa throughnon-covalent interactions. The trimeric Ara h1 structures oftenaggregate, forming multimers of up to 600-700 kDa. Peanut allergenicprotein Ara h2 migrates as a doublet at approximately 20 kDa. Thisdoublet consists of two isoforms that are nearly identical except forthe insertion of the sequence DPYSPS in the higher molecular weightisoform. Peanut allergenic protein Ara h6, was identified as a proteinwith a molecular weight of approximately 15 kDa based on SDS-PAGE and14,981 Da as determined by mass spectroscopy.

According to yet another preferred embodiment of this first aspect ofthe present invention, the present allergen is an allergenic protein,preferably of protein selected from the group consisting of foodproteins or venom proteins.

The present composition preferably comprises pharmaceutically acceptablecarriers, diluents and/or excipients.

Considering the beneficial properties of the present compositions inimmunotherapy, the present invention relates, according to a secondaspect, to a composition comprising an allergen, wherein substantially100% of said allergen in said composition is complexed with aluminum,for use in a therapeutic or prophylactic treatment of a mammal,preferably a human mammal, suffering from an allergy by immunotherapy.

According to an especially preferred embodiment of this second aspect,the allergen is a peanut kernel protein extract, preferably modified byreduction and subsequent alkylation, and the allergy is peanut allergy.

Considering the beneficial properties of the present compositions inimmunotherapy, the present invention relates, according to a thirdaspect, to a composition comprising an allergen, wherein substantially100% of said allergen in said composition is complexed with aluminum,for use in a prophylactic treatment for desentizing the immune system ofa mammal, preferably a human mammal, for said allergen.

According to an especially preferred embodiment of this third aspect,the allergen is a peanut kernel protein extract, preferably modified byreduction and subsequent alkylation, and the immune system isdesensitized for exposure to peanuts.

According to a fourth aspect, the present invention relates to acomposition comprising an allergen, wherein substantially 100% of saidallergen in said composition is complexed with aluminum, for use inmedicine.

According to a fifth aspect, the present invention relates to a methodfor immunotherapy comprising administering to a mammal, preferably ahuman mammal, suffering from an allergy a composition comprising anallergen, wherein substantially 100% of said allergen in saidcomposition is complexed with aluminum, in a sufficient amount andduring sufficient time to reduce, or eliminate, an allergic response ofsaid mammal to said allergen.

A typical sufficient amount will be from about 0.1 ng/kg to 10 mg/kg, 10ng/kg to about 100 μg/kg, or 0.1 μg/kg to 1 μg/kg of the aluminumcomplexed allergen relative to the body weight of the individual towhich it is administered. Often, a treatment will comprise starting withthe administration of dosages at the lower end of these ranges andincreasing the dosages as the treatment progresses.

For desensitization treatment, it is typically necessary for the patientto receive frequent administrations, e.g., initially every one, two orthree days, gradually reducing to once every two or three weeks. Othersuitable desensitization programs include subcutaneous injections onceevery 2-4 weeks the dosage of which injections may gradually increaseover a period of 3-6 months, and then continuing every 2-4 weeks for aperiod of up to about 5 years. It is also possible, particular forsublingual administration, that daily administrations are given.

Desensitization protocols may also comprise a form of treatmentconventionally known in various equivalent alternative forms as rapiddesensitization, rapid allergen immunotherapy, rapid allergenvaccination, and rapid or rush immunotherapy. In broad terms, thisprocedure aims to advance an allergic patient to an immunizing ormaintenance dose of extract (i.e., allergen) by administering a seriesof injections (or via another suitable carrier) of increasing doses ofthe allergen at frequent (e.g. hourly) intervals. If successful, thepatient will exhibit an improved resistance to the allergen, possiblyeven presenting a total non-reactivity to any subsequent allergenexposure.

Various desensitization protocols are known in the art and may forinstance comprise a method of treating a patient having an immediatehypersensitivity to an allergen using an accelerated rapid immunotherapyschedule in combination with a method of pre-treating such patient withprednisone and histamine antagonists prior to receiving the acceleratedimmunotherapy.

According to an especially preferred embodiment of this fifth aspect,the allergen is a peanut kernel protein extract, preferably modified byreduction and subsequent alkylation, and the allergy is peanut allergy.

The present invention will be further detailed in the following examplesdisclosing specifically preferred embodiments of the present invention.In the examples, reference is made to figures wherein:

FIG. 1: shows temperature changes in sensitized mice challenged withdifferent preparations of peanut extract adsorbed to aluminum.Temperature changes were measured for 90 minutes after challenge with0.6 mg/mouse, 0.6 mg/mouse adsorbed to 0.18 mg/ml alum, 0.45 mg/mousealum, 0.9 mg/ml alum or 5.46 mg/ml alum. As a control sensitized micewere challenged with PBS/alum;

FIG. 2: shows symptom scores of sensitized mice challenged withdifferent preparations of peanut extract adsorbed to aluminum. Symptomscores were assigned after challenge on a scale from 0 (no symptoms) to5 (death).

FIG. 3: shows a schematic overview of the time lime used forsensitization and challenge;

FIG. 4: shows the results of an i.p. challenge at day 98 in vivo mousemodel for peanut allergy;

FIG. 5: shows the results of an i.p. challenge at day 112 in vivo mousemodel for peanut allergy;

FIG. 6: shows the results mast protease secretion 1 (mMCP-1) one dayafter challenge in vivo mouse model for peanut allergy;

FIG. 7: shows IgE, IgG1 and IgG2a antibody levels in all groups testedof the in vivo mouse model for peanut allergy.

EXAMPLES Example 1 Complexing of Aluminum and an Allergen SignificantlyIncreases Safety Introduction

This example demonstrates that the complexing of aluminum with anallergen significantly increases safety of a composition used forimmunotherapy. This was demonstrated using an antigenic peanut extractcoupled to different concentrations of aluminum hydroxide. In an in vivomouse model for peanut allergy, these different test preparations wereanalyzed for safety.

Material and Methods Mice

Five-week-old specific pathogen-free female C3H/HeOuJ mice werepurchased from Charles River, France. All mice were housed underspecific pathogen-free conditions within the animal care facility at theUtrecht University, The Netherlands. Experiments were approved by theAnimal Experiments Committee of the Utrecht University. The diet usedcontained vegetable protein (including soy) but was free of peanutproteins.

Test Preparations

Previous studies have demonstrated that a challenge of 0.6 mg peanutextract (PE) per mouse that is not bound to aluminum results in aprofound anaphylactic response. It is also know that a challenge with0.1 mg PE is capable of inducing profound changes in temperature andsymptom score in sensitized mice.

In a pilot study, the relation between the amount of aluminum added andthe percentage of aluminum complexed peanut extract was investigated.For this, different amounts of aluminum hydroxide were added to a sampleof peanut extract (100% protein) and the sample was centrifuged topellet the aluminum complexed peanut extract. Subsequently, thepercentage free protein in the supernatant was determined for assessingthe amount of pelleted, thus aluminum complexed, allergen wherein thepercentage aluminum complexed allergen is 100%—the percentage freeprotein found in the supernatant.

The different aluminum concentrations used in the present example werebased on the binding of ˜100% (5.46 mg/ml) or ˜90% (0.9 mg/ml alum),˜70% (0.45 mg/ml alum), ˜40% (0.18 mg/ml alum) of the total extract. Apositive control (0.6 mg/ml PE without alum) was also included.

Sensitization and Challenge

Mice (n=6) were sensitized by intragastric (i.g.) administration of 6 mgpeanut extract (PE) and 15 μg Cholera Toxin (CT, List BiologicalLaboratories, Inc.) in 400 μl PBS per mouse on days 0, 1, 2, 7, 14, 21,28. Control mice received PBS with 15 μg CT/mouse in 400 μl PBS permouse. On day 42, all groups of mice were subcutaneously (s.c)challenged in the neck with 200 μl of the different test preparations ortheir respective control.

Assessment of Anaphylaxis

As an objective parameter of anaphylactic shock, body temperature wasmeasured by means of rectal thermometry every 10-20 minutes for 90minutes after s.c. challenge. In addition, clinical symptoms were scoredusing a scoring system from 0 (no symptoms) to 5 (death).

Results

The percentage aluminum complexed peanut extract was determined and theresults are summarized in Table 1 below.

TABLE 1 Percentage aluminum complex peanut extract Aluminum hydroxidePercentage Preparation concentration Percentage free complexed per mouse(mg/ml) protein protein 0.6 mg PE 0 100%  0% 0.6 mg PE 0.18  60%  40%0.6 mg PE 0.45  30%  70% 0.6 mg PE 0.9  10%  90% 0.6 mg PE 5.46  0% 100%

A subcutaneous challenge with 0.6 mg non-complexed PE per mouse resulteda severe anaphylactic shock response in all mice as measured by theirtemperature drop (FIG. 1) and clinical symptom score (FIG. 2). When thepeanut preparation was fully (100%) adsorbed to aluminum, none of themice showed any signs of anaphylactic shock symptoms.

Challenging mice with preparations containing different amounts ofaluminum only securing partial adsorption of PE resulted in a delayedresponse in the groups challenged with the preparation containing 60%and 30% non-adsorbed material. In the group challenged with thepreparation containing ˜10% non-adsorbed material there was a delay aswell as a decrease in the response. Non-sensitized mice did not respondto any of the challenges (data not shown) and sensitized mice that werechallenged with aluminum only also showed no response.

Conclusion

Mice that were sensitized and challenged with preparations containingsimilar amounts of PE but varying amounts of alum responded differentlyto a challenge. Complete (100%) binding of PE to aluminum aborted thepotency of the peanut extract whereas partial binding still resulted inan anaphylactic response. The present example clearly shows the potencyof aluminum to prevent mice from suffering an anaphylactic shock.

Example 2 Complexing of Aluminum and an Allergen Significantly Increasesthe Response of the Immune System Resulting to an Increased EfficacyProfile Introduction

This example demonstrates that the complexing of aluminum with anallergen significantly increases the response of the immune systemresulting to an increased efficacy profile. This was demonstrated usingan antigenic peanut extract coupled to aluminum hydroxide. In an in vivomouse model for peanut allergy an immunotherapy, test preparations wereanalyzed for efficacy.

Material and Methods Mice

Five-week-old specific pathogen-free female C3H/HeOuJ mice werepurchased from Charles River, France. All mice were housed underspecific pathogen-free conditions within the animal care facility at theUtrecht University, The Netherlands. Experiments were approved by theAnimal Experiments Committee of the Utrecht University. The diet usedcontained vegetable protein (including soy) but was free of peanutproteins.

Sensitization and Challenge

Mice (n=6 per group) were sensitized by intragastric (i.g.)administration of 6 mg peanut extract (PE) and 15 μg Cholera Toxin (CT,List Biological Laboratories, Inc.) in 400 μl PBS per mouse on days 0,1, 2, 7, 14, 21, 28. Control mice received PBS with 15 μg CT/mouse in400 μl PBS per mouse. From day 42, all groups of mice weresubcutaneously (s.c.) de-sensitized in the neck, twice a week for sixweeks, with 200 μl of the different test preparations or theirrespective control (FIG. 3). The test preparations were tested in aconcentration of 0.1 mg/mouse per injection and were either non-adsorbedor adsorbed to 1.82 mg/ml aluminum.

Assessment of Anaphylaxis

As an objective parameter of anaphylactic shock, body temperature wasmeasured by means of rectal thermometry every 10-20 minutes for 90minutes after i.p. challenge. One day after challenge, blood was takenfor the measurement of antibodies and mMCP-1 (mast cell protease 1). Onday 98 and 112, mice were challenge i.p. and their body temperature wasfollowed for 90 minutes after challenge.

Results

An i.p. challenge was given on day 98 and 112. The data show that on day112 both immunotherapy preparations (peanut extract alone and adsorbedto alum) effectively reduced the anaphylactic response as measured bythe drop in temperature after challenge (FIG. 5).

On day 98, the alum-adsorbed extract showed a greater efficacy comparedto the non-adsorbed extract (FIG. 4) demonstrating that the presence ofalum results in a preparation that reaches efficacy at an earlier timepoint compared to the non-alum preparation.

No differences between the 2 preparations were found in the secretion ofthe mast protease 1 (mMCP-1) in the serum one day after the challenge(FIG. 6). However, immunotherapy with both preparations was capable ofdown-regulating mast cell activation, as the release of mMCP-1 wassignificantly greater in the group of allergic mice that did not receiveimmunotherapy (FIG. 6).

Antibodies (IgE, IgG1 and IgG2a) were determined in the serum of allgroups (FIG. 7). IgE levels were elevated in all groups compared to thenegative control (PBS). The group de-sensitized with the alum-adsorbedpeanut extract showed a trend towards an elevated IgE level (Figure 7A)demonstrating a boost of the immune system after the injection of analum-adsorbed preparation.

Mice that received immunotherapy displayed an increased level of IgG1 inthe serum with comparable levels between the alum-adsorbed andnon-adsorbed extract (FIG. 7B). The increase of IgG2a levels (comparablewith IgG4 in human) is dominated by the group treated with thealum-adsorbed peanut preparation (FIG. 7C).

Conclusion

The adsorption of alum to a peanut extract results in a boost of theimmune system leading to an efficacious treatment at an earlier timepoint. Without wishing to be bound to any theory, this could be due tothe elevated levels of IgG2a (comparable to IgG4 in human) in the serumof these mice.

1. Composition suitable for immunotherapy comprising an allergen,wherein substantially 100% of said allergen in said composition iscomplexed, or conjugated, with aluminum.
 2. Composition according toclaim 1, wherein said allergen is a peanut kernel protein extract. 3.Composition according to claim 2, wherein said peanut kernel proteinextract is modified by reduction and subsequent alkylation. 4.Composition according to claim 2 or claim 3, wherein said peanut kernelprotein extract comprises at least Ara h1, Ara h2 and Ara h6 or Ara h2and Ara h6.
 5. Composition according to claim 1, wherein said allergenis an allergenic protein.
 6. Composition according to claim 5, whereinsaid allergenic protein is selected from the group consisting of foodproteins and venom proteins.
 7. Composition according to any of theclaims 1 to 6, said composition further comprises pharmaceuticallyacceptable carriers, diluents and/or excipients.
 8. Compositioncomprising an allergen, wherein substantially 100% of said allergen insaid composition is complexed, or conjugated, with aluminum, for use inthe treatment of a mammal suffering from an allergy by immunotherapy. 9.Use according to claim 8, wherein said mammal is a human mammal.
 10. Useaccording to claim 8 or claim 9, wherein said allergen is a peanutkernel protein extract and said allergy is peanut allergy.
 11. Useaccording to claim 10, wherein said peanut kernel protein extract ismodified by reduction and subsequent alkylation.
 12. Compositioncomprising an allergen, wherein substantially 100% of said allergen insaid composition is complexed, or conjugated, with aluminum, for use ina therapeutic treatment for desentizing the immune system of a mammalfor said allergen or the use in a prophylactic treatment of a mammalwith high predisposition for developing an allergy upon contact withsaid allergen.
 13. Use according to claim 12, wherein said mammal is ahuman mammal.
 14. Use according to claim 12 or claim 13, wherein saidallergen is a peanut kernel protein extract and the immune system isdesensitized for exposure to peanuts.
 15. Use according to claim 14,wherein said peanut kernel protein extract is modified by reduction andsubsequent alkylation.
 16. Composition comprising an allergen, whereinsubstantially 100% of said allergen in said composition is complexed, orconjugated, with aluminum, for use in medicine.
 17. Method forimmunotherapy comprising administering to a mammal suffering from anallergy, or a mammal at risk of developing an allergy, a compositioncomprising an allergen, wherein substantially 100% of said allergen insaid composition is complexed, or conjugated, with aluminum, in asufficient amount and during sufficient time to reduce, or eliminate, anallergic response of said mammal to said allergen.
 18. Method accordingto claim 17, wherein said mammal is a human mammal.
 19. Method accordingto claim 17 or claim 18, wherein said allergen is a peanut kernelprotein extract and the allergic response is an allergic response topeanut exposure.
 20. Method according to claim 19, wherein said peanutkernel protein extract is modified by reduction and subsequentalkylation.